Is epithelial-mesenchymal transition related to the biological behavior of salivary gland neoplasms?

OBJECTIVE: To evaluate and compare the expression of E-cadherin, Snail1 and Twist1 in pleomorphic adenomas (PAs), adenoid cystic carcinomas (AdCCa) and carcinoma ex-pleomorphic adenomas (CaexPA) of salivary glands, as well as investigate possible associations with clinicopathological parameters. STUDY DESIGN: E-cadherin, Snail1 and Twist1 antibody immunostaining were analyzed semiquantitatively in 20 PAs, 20 AdCCas and 10 CaexPAs. Cases were classified as low and high expression for analysis of the association with clinicopathological parameters. RESULTS: Compared to PAs, AdCCas and CaexPAs exhibited higher nuclear expression of Snail1 (p = 0.021 and p = 0.028, respectively) and Twist1 (p = 0.009 and p = 0.001). Membranous and cytoplasmic expression of E-cadherin were positively correlated in PAs, AdCCas and CaexPAs (r = 0.645, p = 0.002; r = 0.824, p < 0.001; r = 0.677, p = 0.031). In PAs, positive correlation was found between nuclear expression of Snail1 and membrane expression of E-cadherin (r = 0.634; p = 0.003), as well as between nuclear expression of Snail1 and Twist1 (r = 0.580; p = 0.007). Negative correlations were detected between membrane expression of E-cadherin and cytoplasmic expression of Snail1 in AdCCas (r = - 0.489; p = 0.029). CONCLUSIONS: E-cadherin, Twist1, and Snail1 may participate in modulating events related to cell differentiation and adhesion in PAs and to biological behavior in AdCCas and CaexPAs, which indicates the involvement of EMT in these processes. Furthermore, the expression of these proteins in these carcinomas may reflect the plasticity feature of EMT.

Collagen-alginate 3D microscaffolds for studying cellular migration.

Metastasis is one of the major causes for cancer mortality. Its early steps comprise of invasion of basement membrane and migration. Thus, it is hypothesized that a platform, that allows quantification and grading of migration capability of cells can potentially be used for predicting metastatic potential. Two-dimensional (2D) models have been rendered inadequate for modelling in-vivo microenvironment due to various reasons. To attenuate homogeneity observed in 2D, three-dimensional (3D) platforms supplemented with bioinspired components have been designed. Unfortunately, till date there are no simple models to capture the migration of cells in 3D along with quantification of the process. In this study, we report an alginate-collagen based 3D model system, which can predict the migratory property of the cells within 72 h. The micron size of the scaffold enabled faster readout and the optimum pore-size provided conducive cellular growth environment. The platform's ability to allow observation of cellular migration was validated by encapsulating cells with transiently upregulated matrix metalloprotease 9 (MMP9), which has been reported to play a significant role in migration of cells during metastasis. The readout for migration was clustering of cells in the microscaffolds detected in a short span of 48 h. The observed clustering in MMP9 upregulated cells was validated by observing changes in the epithelial-mesenchymal transition (EMT) markers. Thus, this simple 3D platform can be used to study migration and predict the metastatic potential of cells.

NEIL3 promotes hepatoma epithelial-mesenchymal transition by activating the BRAF/MEK/ERK/TWIST signaling pathway.

Hepatocellular carcinoma (HCC) is among the most prevalent visceral neoplasms. So far, reliable biomarkers for predicting HCC recurrence in patients undergoing surgery are far from adequate. In the aim of searching for genetic biomarkers involved in HCC development, we performed analyses of cDNA microarrays and found that the DNA repair gene NEIL3 was remarkably overexpressed in tumors. NEIL3 belongs to the Fpg/Nei protein superfamily, which contains DNA glycosylase activity required for the base excision repair for DNA lesions. Notably, the other Fpg/Nei family proteins NEIL1 and NEIL2, which have the same glycosylase activity as NEIL3, were not elevated in HCC; NEIL3 was specifically induced to participate in HCC development independently of its glycosylase activity. Using RNA-seq and invasion/migration assays, we found that NEIL3 elevated the expression of epithelial-mesenchymal transition (EMT) factors, including the E/N-cadherin switch and the transcription of MMP genes, and promoted the invasion, migration, and stemness phenotypes of HCC cells. Moreover, NEIL3 directly interacted with the key EMT player TWIST1 to enhance invasion and migration activities. In mouse orthotopic HCC studies, NEIL3 overexpression also caused a prominent E-cadherin decrease, tumor volume increase, and lung metastasis, indicating that NEIL3 led to EMT and tumor metastasis in mice. We further found that NEIL3 induced the transcription of MDR1 (ABCB1) and BRAF genes through the canonical E-box (CANNTG) promoter region, which the TWIST1 transcription factor recognizes and binds to, leading to the BRAF/MEK/ERK pathway-mediated cell proliferation as well as anti-cancer drug resistance, respectively. In the HCC cohort, the tumor NEIL3 level demonstrated a high positive correlation with disease-free and overall survival after surgery. In conclusion, NEIL3 activated the BRAF/MEK/ERK/TWIST pathway-mediated EMT and therapeutic resistances, leading to HCC progression. Targeted inhibition of NEIL3 in HCC individuals with NEIL3 induction is a promising therapeutic approach. © 2022 The Pathological Society of Great Britain and Ireland.

The Post-Translational Regulation of Epithelial-Mesenchymal Transition-Inducing Transcription Factors in Cancer Metastasis.

Epithelial-mesenchymal transition (EMT) is generally observed in normal embryogenesis and wound healing. However, this process can occur in cancer cells and lead to metastasis. The contribution of EMT in both development and pathology has been studied widely. This transition requires the up- and down-regulation of specific proteins, both of which are regulated by EMT-inducing transcription factors (EMT-TFs), mainly represented by the families of Snail, Twist, and ZEB proteins. This review highlights the roles of key EMT-TFs and their post-translational regulation in cancer metastasis.

Twist3 is required for dedifferentiation during extraocular muscle regeneration in adult zebrafish.

Severely damaged adult zebrafish extraocular muscles (EOMs) regenerate through dedifferentiation of residual myocytes involving a muscle-to-mesenchyme transition. Members of the Twist family of basic helix-loop-helix transcription factors (TFs) are key regulators of the epithelial-mesenchymal transition (EMT) and are also involved in craniofacial development in humans and animal models. During zebrafish embryogenesis, twist family members (twist1a, twist1b, twist2, and twist3) function to regulate craniofacial skeletal development. Because of their roles as master regulators of stem cell biology, we hypothesized that twist TFs regulate adult EOM repair and regeneration. In this study, utilizing an adult zebrafish EOM regeneration model, we demonstrate that inhibiting twist3 function using translation-blocking morpholino oligonucleotides (MOs) impairs muscle regeneration by reducing myocyte dedifferentiation and proliferation in the regenerating muscle. This supports our hypothesis that twist TFs are involved in the early steps of dedifferentiation and highlights the importance of twist3 during EOM regeneration.

Constitutive Expression of NRAS with Q61R Driver Mutation Activates Processes of Epithelial-Mesenchymal Transition and Leads to Substantial Transcriptome Change of Nthy-ori 3-1 Thyroid Epithelial Cells.

The Q61R mutation of the NRAS gene is one of the most frequent driver mutations of thyroid cancer. Tumors with this mutation are characterized by invasion into blood vessels and formation of distant metastases. To study the role of this mutation in the growth of thyroid cancer, we developed a model system on the basis of thyroid epithelial cell line Nthy-ori 3-1 transduced by a lentiviral vector containing the NRAS gene with the Q61R mutation. It was found that the expression of NRAS(Q61R) in thyroid epithelial cells has a profound influence on groups of genes involved in the formation of intercellular contacts, as well as in processes of epithelial-mesenchymal transition and cell invasion. The alteration in the expression of these genes affects the phenotype of the model cells, which acquire traits of mesenchymal cells and demonstrate increased ability for survival and growth without attachment to the substrate. The key regulators of these processes are transcription factors belonging to families SNAIL, ZEB, and TWIST, and in different types of tumors the contribution of each individual factor can vary greatly. In our model system, phenotype change correlates with an increase in the expression of SNAIL2 and TWIST2 factors, which indicates their possible role in regulating invasive growth of thyroid cancer with the mutation of NRAS(Q61R).

Endothelial-specific deletion of Ets-1 attenuates Angiotensin II-induced cardiac fibrosis via suppression of endothelial-to-mesenchymal transition.

Cardiac fibrosis is a common feature in chronic hypertension patients with advanced heart failure, and endothelial-tomesenchymal transition (EndMT) is known to promote Angiotensin II (Ang II)-mediated cardiac fibrosis. Previous studies have suggested a potential role for the transcription factor, ETS-1, in Ang II-mediated cardiac remodeling, however the mechanism are not well defined. In this study, we found that mice with endothelial Ets-1 deletion showed reduced cardiac fibrosis and hypertrophy following Ang II infusion. The reduced cardiac fibrosis was accompanied by decreased expression of fibrotic matrix genes, reduced EndMT with decreased Snail, Slug, Twist, and ZEB1 expression, as well as reduced cardiac hypertrophy and expression of hypertrophyassociated genes was observed. In vitro studies using cultured H5V cells further confirmed that ETS-1 knockdown inhibited TGF-ß1-induced EndMT. This study revealed that deletion of endothelial Ets-1 attenuated Ang II-induced cardiac fibrosis via inhibition of EndMT, indicating an important ETS-1 function in mediating EndMT. Inhibition of ETS-1 could be a potential therapeutic strategy for treatment of heart failure secondary to chronic hypertension. [BMB Reports 2019; 52(10): 595-600].

High Glucose Promotes Epithelial-Mesenchymal Transition of Uterus Endometrial Cancer Cells by Increasing ER/GLUT4-Mediated VEGF Secretion.

BACKGROUND/AIMS: Uterus endometrial cancer (UEC) is the common malignancy among gynecologic cancers, and most of them are type I estrogen-dependent UEC. Diabetes is well-known risk factor for the development of UEC. However, the underlying link between high glucose (HG) and the estrogen receptor in UEC remains unclear. Epithelial-mesenchymal transition (EMT) has also been shown to occur during the initiation of metastasis in cancer progression. The aim of this study was to determine the relationships and roles of HG, estrogen receptor and EMT in the growth and migration of UEC. METHODS: The expression of glucose transport protein 4 (GLUT4) in the control endometrium and UEC tissues was detected by immunohistochemistry (IHC); the cell viability and invasion were analyzed through CCK-8 and Matrigel invasion assays; the transcriptional level of EMT-related genes was evaluated through real-time PCR; and the effect of HG and / or GLUT4 on estrogen receptors, vascular endothelial growth factor (VEGF) and its receptor VEGFR was analyzed through western blotting, ELISA and flow cytometry (FCM) assay, respectively. In addition, Ishikawa-xenografted nude mice were constructed and were used to analyze the effect of estrogen and GLUT4 on the growth of UEC in vivo. RESULTS: Here, we found that exposure to HG led to a high level of viability and invasion of UEC cell lines (UECC, Ishikawa and RL95-2 cells). Compared with the normal endometrium, a higher level of GLUT4 was observed in UEC tissues. Silencing GLUT4 obviously inhibited the HG-promoted viability, invasion and expression of EMT-related genes (TWIST, SNAIL and CTNNB1) of UECC promoted by HG. Further analysis showed that HG and GLUT4 promoted the secretion of VEGF and expression of VEGFR in UECC. Treatment with HG led to the increase of estrogen receptor α (ERα) and ß (ERß) in UECC, blocking ERα or ERß resulted in the decreases in GLUT4 expression, TWIST, SNAIL and CTNNB1 transcription, and VEGF and VEGFR expression in UECC. Treatment with anti-human VEGF neutralizing antibody restricted the viability and invasion of UECC that was induced by HG and estrogen. Exposure to estrogen accelerated growth, VEGF production, and TWIST and CTNNB1 expression in UEC in Ishikawa-xenografted nude mice, and silencing GLUT4 restricted these effects. CONCLUSION: These data suggest that HG increases GLUT4 and VEGF/VEGFR expression, further promotes EMT process and accelerates the development of UEC by up-regulating ER.

Apoptotic cell induction of miR-10b in macrophages contributes to advanced atherosclerosis progression in ApoE-/- mice.

Aims: We previously found that miR-10b inhibits cholesterol efflux from thioglycollate-elicited mouse peritoneal macrophages through repressing ATP binding cassette transporter (ABCA1). Herein, we deciphered the mechanism underlying macrophage miR-10b expression and the role of miR-10b in atherosclerosis. Methods and results: MiR-10b expression was increased in the arteries with advanced but not early atherosclerotic plaques of ApoE-/- mice. Free cholesterol-induced macrophage apoptotic cells (FC-AM) promoted miR-10b expression in mouse resident peritoneal macrophages (RPM) by up-regulation of Twist1/2 in a Mer receptor tyrosine kinase dependent manner. Surprisingly, antagomiR-10b de-repressed ABCA1 in RPM engulfing FC-AM but not in RPM alone or RPM-derived foam cells; systemic delivery of antagomiR-10b enhanced reverse cholesterol transport from RPM engulfing FC-AM but not from RPM or the foam cells in ApoE-/- mice. Mechanistically, RPM engulfing FC-AM possessed sufficient miR-10b functionally repressing ABCA1 expression, whereas RPM and foam cells had little miR-10b incompetently repressing ABCA1 expression. Notably, antagomiR-10b administration reduced advanced plaque size and also enhanced plaque stability in ApoE-/-mice, which were associated with increased plaque macrophage ABCA1 expression and reduced plaque apoptosis and inflammation. However, antagomiR-10b administration did not affect early atherosclerotic plaque formation in ApoE-/- mice. Conclusion: These data suggest that apoptotic cell induction of miR-10b in macrophages is important in advanced atherosclerosis progression.

Hyaluronic acid conjugated nanoparticle delivery of siRNA against TWIST reduces tumor burden and enhances sensitivity to cisplatin in ovarian cancer.

TWIST protein is critical to development and is activated in many cancers. TWIST regulates epithelial-mesenchymal transition, and is linked to angiogenesis, metastasis, cancer stem cell phenotype, and drug resistance. The majority of epithelial ovarian cancer (EOC) patients with metastatic disease respond well to first-line chemotherapy but most relapse with disease that is both metastatic and drug resistant, leading to a five-year survival rate under 20%. We are investigating the role of TWIST in mediating these relapses. We demonstrate TWIST-siRNA (siTWIST) and a novel nanoparticle delivery platform to reverse chemoresistance in an EOC model. Hyaluronic-acid conjugated mesoporous silica nanoparticles (MSN-HAs) carried siTWIST into target cells and led to sustained TWIST knockdown in vitro. Mice treated with siTWIST-MSN-HA and cisplatin exhibited specific tumor targeting and reduction of tumor burden. This platform has potential application for overcoming clinical challenges of tumor cell targeting, metastasis and chemoresistance in ovarian and other TWIST overexpressing cancers.

Deubiquitinase USP9X promotes cell migration, invasion and inhibits apoptosis of human pancreatic cancer.

Ubiquitin specific peptidase 9, X-linked (USP9X), a significant regulatory protease in protein ubiquitination, has been proven to act as a proto-oncogene in several types of cancers, such as cervix, colon, breast, brain and lung cancers. The prognosis of patients with pancreatic ductal adenocarcinoma (PDAC) is extremely poor due to its high invasive and metastatic abilities. Nevertheless, whether USP9X acts as a proto-oncogene or a tumor-suppressor gene in PDAC is controversial and the mechanism of metastasis remains unknown. The present study focused on the effect of USP9X on the migration, invasion and apoptosis of PADC cells. We analyzed the expression of USP9X in pancreatic cancer tissues of different pathologic grades by immunohistochemical analysis. USP9X expression in the pancreatic cancer tissues was markedly increased in contrast to that noted in the adjacent non-tumor tissues. USP9X expression in PANC-1 cells was downregulated after transfection of shRNA-USP9X. Knockdown of endogenous USP9X expression evidently inhibited the migration and invasion of PANC-1 cells, and promoted cell apoptosis. Meanwhile, expression levels of Snail, Twist, N-cadherin and vimentin were downregulated. E-cadherin expression was negatively correlated with USP9X expression and the expression of survivin was also downregulated in the PANC-1 cells. In brief, USP9X promoted the migration and invasion of PANC-1 cells probably by provoking epithelial-mesenchymal transition, and also inhibited apoptosis. We believe that USP9X is a major oncogene that may play a significant role in the treatment and prognosis of PDAC.

Epithelial-mesenchymal transition of ovarian cancer cells is sustained by Rac1 through simultaneous activation of MEK1/2 and Src signaling pathways.

Epithelial-mesenchymal transition (EMT) is regarded as a crucial contributing factor to cancer progression. Diverse factors have been identified as potent EMT inducers in ovarian cancer. However, molecular mechanism sustaining EMT of ovarian cancer cells remains elusive. Here we show that the presence of SOS1/EPS8/ABI1 complex is critical for sustained EMT traits of ovarian cancer cells. Consistent with the role of SOS1/EPS8/ABI1 complex as a Rac1-specific guanine nucleotide exchange factor, depleting Rac1 results in the loss of most of mesenchymal traits in mesenchymal-like ovarian cancer cells, whereas expressing constitutively active Rac1 leads to EMT in epithelial-like ovarian cancer cells. With the aid of clinically tested inhibitors targeting various EMT-associated signaling pathways, we show that only combined treatment of mitogen-activated extracellular signal-regulated kinase 1/2 (MEK1/2) and Src inhibitors can abolish constitutively active Rac1-led EMT and mesenchymal traits displayed by mesenchymal-like ovarian cancer cells. Further experiments also reveal that EMT can be induced in epithelial-like ovarian cancer cells by co-expressing constitutively active MEK1 and Src rather than either alone. As the activities of Erk and Src are higher in ovarian cancer cells with constitutively active Rac1, we conclude that Rac1 sustains ovarian cancer cell EMT through simultaneous activation of MEK1/2 and Src signaling pathways. Importantly, we demonstrate that combined use of MEK1/2 and Src inhibitors effectively suppresses development of intraperitoneal xenografts and prolongs the survival of ovarian cancer-bearing mice. This study suggests that cocktail of MEK1/2 and Src inhibitors represents an effective therapeutic strategy against ovarian cancer progression.

The Morphogenesis of Cranial Sutures in Zebrafish.

Using morphological, histological, and TEM analyses of the cranium, we provide a detailed description of bone and suture growth in zebrafish. Based on expression patterns and localization, we identified osteoblasts at different degrees of maturation. Our data confirm that, unlike in humans, zebrafish cranial sutures maintain lifelong patency to sustain skull growth. The cranial vault develops in a coordinated manner resulting in a structure that protects the brain. The zebrafish cranial roof parallels that of higher vertebrates and contains five major bones: one pair of frontal bones, one pair of parietal bones, and the supraoccipital bone. Parietal and frontal bones are formed by intramembranous ossification within a layer of mesenchyme positioned between the dermal mesenchyme and meninges surrounding the brain. The supraoccipital bone has an endochondral origin. Cranial bones are separated by connective tissue with a distinctive architecture of osteogenic cells and collagen fibrils. Here we show RNA in situ hybridization for col1a1a, col2a1a, col10a1, bglap/osteocalcin, fgfr1a, fgfr1b, fgfr2, fgfr3, foxq1, twist2, twist3, runx2a, runx2b, sp7/osterix, and spp1/ osteopontin, indicating that the expression of genes involved in suture development in mammals is preserved in zebrafish. We also present methods for examining the cranium and its sutures, which permit the study of the mechanisms involved in suture patency as well as their pathological obliteration. The model we develop has implications for the study of human disorders, including craniosynostosis, which affects 1 in 2,500 live births.

Quercetin Inhibits Fibroblast Activation and Kidney Fibrosis Involving the Suppression of Mammalian Target of Rapamycin and ß-catenin Signaling.

Quercetin, a flavonoid found in a wide variety of plants and presented in human diet, displays promising potential in preventing kidney fibroblast activation. However, whether quercetin can ameliorate kidney fibrosis in mice with obstructive nephropathy and the underlying mechanisms remain to be further elucidated. In this study, we found that administration of quercetin could largely ameliorate kidney interstitial fibrosis and macrophage accumulation in the kidneys with obstructive nephropathy. MTORC1, mTORC2, ß-catenin as well as Smad signaling were activated in the obstructive kidneys, whereas quercetin could markedly reduce their abundance except Smad3 phosphorylation. In cultured NRK-49F cells, quercetin could inhibit α-SMA and fibronectin (FN) expression induced by TGFß1 treatment. MTORC1, mTORC2, ß-catenin and Smad signaling pathways were stimulated by TGFß1 at a time dependent manner. Similar to those findings in the obstructive kidneys, mTORC1, mTORC2 and ß-catenin, but not Smad signaling pathways were remarkably blocked by quercetin treatment. Together, these results suggest that quercetin inhibits fibroblast activation and kidney fibrosis involving a combined inhibition of mTOR and ß-catenin signaling transduction, which may act as a therapeutic candidate for patients with chronic kidney diseases.

Twist-mediated Epithelial-mesenchymal Transition Promotes Breast Tumor Cell Invasion via Inhibition of Hippo Pathway.

Twist is a key transcription factor for Epithelial-mesenchymal transition (EMT), which is a cellular de-differentiation program that promotes invasion and metastasis, confers tumor cells with cancer stem cell (CSC)-like characteristics, and increases therapeutic resistance. However, the mechanisms that facilitate the functions of Twist remain unclear. Here we report that Twist overexpression increased expression of PAR1, an upstream regulator of the Hippo pathway; PAR1 promotes invasion, migration, and CSC-like properties in breast cancer by activating the transcriptional co-activator TAZ. Our study indicates that Hippo pathway inhibition is required for the increased migratory and invasiveness ability of breast cancer cells in Twist-mediated EMT.

Expression profile of microRNA-200 family in cholangiocarcinoma arising from choledochal cyst.

BACKGROUND AND AIM: The risk of cholangiocarcinoma (cCC) arising from choledochal cyst (CC-CC) is imminent, if the latter not treated appropriately in time. Epithelial-to-mesenchymal transition (EMT) is considered a critical step for various solid cancers, which is regulated by the microRNA-200 (miR-200) family. The aim of this study was to assess the role of miR-200 family in the pathogenesis of CC-CC. METHODS: Sixteen patients with CC-CC were enrolled and 254 patients with conventional cCC served as clinicopathologic controls. Fifty-four cCC were selected to compare the miR-200 family expression and immunohistochemical characteristics. Gain-and loss-of-function studies of miR-200 family were conducted using the cCC cell lines. RESULTS: CC-CC were younger (P < 0.01), more female- predominated (P < 0.01), and rarely associated with lithiasis (P < 0.01) compared with those of cCC. miR-200 family was down-regulated in CC-CC, while miR-200 family was paradoxically up-regulated in cCC (P < 0.01). CC-CC exhibited overt overexpression of mesenchymal markers including ZEB1, Twist, Snail, and vimentin as well an aberrant E-cadherin expression in comparison with cCC. In vitro migration assay showed that cCC cells bearing lower miR-200 s levels exhibited stronger migration ability. Invasive ability of cCC cells was increased after miR-200 s knockdown, accompanied by up-regulation of mesenchymal markers. CONCLUSIONS: CC-CC was characterized by distinct demographics, precipitating factors, and down-regulation of miR-200 family, compared with those of cCC. The pathogenesis of CC-CC might partly link to the silencing of miR-200 family, acting via ZEB1-directed EMT activation.